Abstract

In response to glucose, mouse β-cells display slow oscillations of the membrane potential and cytosolic free Ca2+ concentration ([Ca2+]i), whereas rat β-cells display a staircase increase in these parameters. Mouse and rat islet cells differ also by their level
of Na/Ca exchanger (NCX) activity. The view that the inward current generated by Na/Ca exchange shapes stimulus-induced electrical
activity and [Ca2+]i oscillations in pancreatic β-cells was examined in insulin-producing BRIN-BD11 cells overexpressing the Na/Ca exchanger.
BRIN-BD11 cells were stably transfected with NCX1.7, one of the exchanger isoforms identified in the β-cell. Overexpression
could be assessed at the mRNA and protein level. Appropriate targeting to the plasma membrane could be assessed by microfluorescence
and the increase in Na/Ca exchange activity. In response to K+, overexpressing cells showed a more rapid increase in [Ca2+]i on membrane depolarization as well as a more rapid decrease of [Ca2+]i on membrane repolarization. In response to glucose and tolbutamide, control BRIN cells showed large amplitude [Ca2+]i oscillations. In contrast, overexpressing cells showed a staircase increase in [Ca2+]i without such large oscillations. Diazoxide-induced membrane hyperpolarization restored large amplitude [Ca2+]i oscillations in overexpressing cells. The present data confirm that Na/Ca exchange plays a significant role in the rat β-cell
[Ca2+]i homeostasis, the exchanger being a versatile system allowing both Ca2+ entry and outflow. Our data suggest that the current generated by the exchanger shapes stimulus-induced membrane potential
and [Ca2+]i oscillations in insulin-secreting cells, with the difference in electrical activity and [Ca2+]i behavior seen in mouse and rat β-cells resulting in part from a difference in Na/Ca exchange activity between these two cells.

Ca2+ plays a key role in the process of glucose-induced insulin release from the pancreatic β-cell. When the β-cell is exposed
to glucose, a complex series of events is initiated that culminate in a rise in cytosolic free Ca2+ concentration ([Ca2+]i), which triggers the exocytosis of insulin (1–4).

Like most other cells, the β-cell is equipped with a double system responsible for Ca2+ extrusion: a plasma membrane Ca2+-ATPase and a Na/Ca exchange transport system (5,6). The Na/Ca exchanger is an electrogenic transporter located at the plasma membrane that couples the exchange of Na+ and Ca2+ with a stoichiometry of three Na+ for one Ca2+ (7). In the cardiac myocyte, Na/Ca exchange is the major mechanism of Ca2+ extrusion, restoring basal Ca2+ levels between heartbeats (8). The exchanger may also reverse during the heart cycle and hence allow Ca2+ entry during the systole (9). In the rat pancreatic β-cell, Na/Ca exchange displays a quite high capacity (10) and participates in the control of [Ca2+]i and insulin release (11–13).

The Na/Ca exchanger (NCX) was cloned from dog heart 10 years ago (14). Up to now, three mammalian isoforms of NCX have been cloned: NCX1 (14), NCX2 (15), and NCX3 (16), representing the products of three distinct genes. Although NCX1 is widely distributed in various tissues, NCX2 and NCX3
seem to be restricted to brain and skeletal muscle (15,16). Several splice variants of NCX1 and NCX3 have been described, each exhibiting a specific tissue distribution (17,18). Sequence analysis of the intron-exon boundaries in the alternative splicing region of NCX1 revealed the presence of two
mutually exclusive exons (A and B) and four cassette exons (C, D, E, and F) (17–19). In the heart, only one NCX1 splice variant appears to be present (NCX1.1), whereas in other tissues, two or even three
variants (eye) are expressed (17,19). Rat pancreatic islet cells, purified β-cells, and RINm5F cells express two NCX1 splice variants (NCX1.3 and NCX1.7) but
do not express NCX2 (20).

Differences in Na/Ca exchange activity appear to exist between rat and mouse β-cells. Indeed, in the rat β-cell, Na/Ca exchange
appears to be responsible for up to 70% of Ca2+ removal from the cytoplasm upon membrane repolarization (13), whereas in the mouse, the exchanger contributes to Ca2+ removal only at [Ca2+]i >1 μmol/l, where it accounts for a maximum of 35% of the total removal rate (21). Moreover, Na/Ca exchange was shown to mediate Ca2+ entry (through reverse Na/Ca exchange) in response to membrane depolarization in rat pancreatic β-cells (13), whereas this does not appear to be the case in the mouse pancreatic β-cell (21). Thus, although the mouse β-cell Na/Ca exchanger is membrane potential-dependent and may thus reverse when its reversal
potential is overrun, it does not appear to reverse under physiological conditions (21). These species-specific differences in activity of the exchanger could result from different levels of Na/Ca exchanger expression,
as suggested by a 50% lower mRNA transcription in mouse compared with rat pancreatic islets (22). In addition, the splice variants expressed in rat and mouse islets are not the same, leaving the possibility of a differential
regulation of the exchanger in these two species (22).

In the presence of insulin-releasing glucose concentrations (11.1 mmol/l), the mouse pancreatic β-cell generates a characteristic
pattern of electrical activity that consists of slow oscillations between a depolarized plateau potential, on which action
potentials are superimposed (equaling bursts), and repolarized electrically silent intervals (23). In contrast, the rat β-cell displays rapid depolarizations to a plateau accompanied by the firing of low- amplitude action
potentials (24). Likewise, in the presence of 11.1 mmol/l glucose, mouse islets show [Ca2+]i oscillations that match the oscillations of the membrane potential (25) and insulin release (26,27). In contrast, rat islets display, according to some studies, either a dose-dependent increase in [Ca2+]i with no detectable oscillations (24) or an initial and transient rise in [Ca2+]i followed by an almost complete return of [Ca2+]i to baseline, with oscillations being only scarcely observed (28). In addition, dispersed β-cells of mice and rats display comparable differences. Thus, single mouse cells display slow oscillations
(0.2–0.5/min) starting from the basal level and onto which faster oscillations are superimposed (2–5/min) (29). In contrast, rat β-cells display more heterogeneous changes in [Ca2+]i, with the majority of cells showing a biphasic increase in [Ca2+]i on which small oscillations are superimposed (13).

In mouse islets, we recently demonstrated that Na/Ca exchange was electrogenic and generated an inward current when functioning
in its forward mode (Ca2+ extrusion) (21). We also observed that extracellular Na+ removal reduced the burst duration, suggesting that the inward current generated by the exchange contributed to the duration
of the bursts (21). Mathematical simulations implementing two existing models for bursting in pancreatic β-cells confirmed that the presence
of Na/Ca exchange activity could substantially increase the plateau fraction of the bursting electrical activity (30) and hence suppress the oscillations of membrane potential and [Ca2+]i. Therefore, the difference in electrical activity and [Ca2+]i oscillations between rat and mouse β-cells could result from a difference in Na/Ca exchange activity. To test this hypothesis,
we stably overexpressed the Na/Ca exchanger (NCX1.7) in BRIN-BD11 cells, an insulin-secreting cell line produced by electrofusion
of normal rat pancreatic β-cell with RINm5F cells (31). The data show that, whereas nontransfected cells responded to glucose and tolbutamide by displaying large amplitude [Ca2+]i oscillations, the transfected cells displayed a staircase increase in [Ca2+]i without such large oscillations. Hence, the current generated by the exchanger appears to shape stimulus-induced membrane
potential and [Ca2+]i oscillations in insulin-secreting cells, and the difference in electrical activity and [Ca2+]i behavior seen in mouse and rat β-cells results in part from a difference in Na/Ca exchange activity.

Cells were plated at 3 × 105 on a 35-mm-diameter plastic dish and incubated 24 h before transfection. BRIN-BD11 cells were then stably transfected with
the mammalian expression vector pcDNA3(−) containing a 2.9-kb insert encoding for human pancreatic β-cell NCX1.7 by the use
of LipofectAMINE (Life Technologies). Positive clones were selected through resistance against G418 (250 μg/ml; Life Technologies)
and verified by RT-PCR, Western blot analysis, immunofluorescence, and Na/Ca exchange activity.

RT-PCR.

For RT-PCR, primers were designed to anneal to conserved sequences flanking the putative splicing area of NCX1. The sense
primer 5′-CATTGGCATCATGGAGGT-3′ and the antisense primer 5′-TTTGCTGGTCAGTGGCTGCTTGTC-3′ correspond to nucleotides 1647–1664
and 2044–2067, respectively, based on rat heart sequence (GenBank accession no. x68191). The oligonucleotides were synthesized
using the phosphoramidite method performed on an Applied Biosystems 394 synthesizer (Perkin Elmer, Zaventem, Belgium).

Total RNA was isolated from control and transfected BRIN-BD11 cells using the RNA Now method (Biogentex, Seabrooke, TX). RNA
(1 μg) was reverse transcribed for 20 min at 42°C and 40 min at 37°C using a Superscript II kit (Life Technologies), with
25 μg/ml oligo(dT) primer and 2.5 μg/ml random primer (Promega), and triphosphate nucleosides (0.5 mmol/l each; Boehringer
Mannheim, Brussels), in the buffer supplied by the manufacturer, in a total volume of 20 μl. RNA complementary to cDNA was
removed using two units of Escherichia coli Rnase H (Boehringer Mannheim) for 20 min at 37°C. The medium was then diluted with 30 μl of 16 mmol/l EDTA, and the reaction
was terminated by heating to 99°C for 5 min. PCR amplification was carried out as described elsewhere (20).

Preparation of BRIN-BD11 plasma membranes.

Plasma membranes were isolated from control BRIN-BD11 cells as well as transfected cells as previously described (22).

45Ca uptake.

For 45Ca uptake, the media used to incubate BRIN-BD11 cells consisted in a Krebs-Ringer HEPES-buffered solution (pH 7.4, 37°C) with
the following composition (in mmol/l): 135 NaCl, 1 CaCl2, 5 KCl, 1 MgCl2, and 10 HEPES/NaOH. Unless otherwise stated, the media were gassed with ambient air. In some experiments, NaCl was iso-osmotically
replaced by sucrose (241 mmol/l; Merck, Darmstadt, Germany) and HEPES/NaOH by HEPES/KOH. For the measurement of Na+i [intracellular Na+]-dependent 45Ca uptake, the different media also contained glucose (2.8 mmol/l; Merck) and nifedipine (5 μmol/l; Calbiochem, La Jolla,
CA). For the measurement of the effect of glucose, the media contained glucose (1.1 or 11.1 mmol/l) without nifedipine. The
method used for the measurement of 45Ca uptake has been described previously (10).

Cytosolic Ca2+concentration measurements.

For the measurement of [Ca2+]i, a drop of the cell suspension (20 μl) containing 50,000 cells was aliquoted onto round glass coverslips placed in Petri
dishes and incubated at 37°C in a 5% CO2/95% O2 incubator. After 2 h, fresh culture medium was added to the Petri dishes. The cells were further incubated for 24 h at 37°C
before use. The medium used to incubate or perfuse the islet cells consisted in a Krebs-Ringer bicarbonate-buffered solution
(pH 7.4, 37°C) having the following composition (in mmol/l): 115 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 24 NaHCO3, and 1.1 glucose. The medium was equilibrated against a mixture of O2 (95%) and CO2 (5%) at pH 7.4. For Na+-free solutions, NaCl was iso-osmotically replaced by choline chloride and NaHCO3 by choline bicarbonate. To avoid cholinergic effects, the latter media contained atropine (10 μmol/l), which was also present
in appropriate control solutions. The different media also contained, when required, 11.1 mmol/l glucose, 50 mmol/l KCl, 100
μmol/l tolbutamide, and 5 μmol/l diazoxide. Stock solution containing fura-2-AM was prepared in dimethyl sulfoxide. Cells
were incubated with fura-2-AM ester (final concentration 2 μmol/l) during 60 min at 37°C in a Krebs-Ringer bicarbonate-buffered
solution containing 1.1 mmol/l glucose. The coverslips were then transferred to a tissue chamber mounted on an inverted fluorescence
microscope (Diaphot TDM; Nikon, Tokyo) for epifluorescence, as described previously (13).

Electrophysiology.

Cells were plated on plastic dishes and used 24 h later. Whole-cell membrane currents were recorded at room temperature (22°C)
with borosilicate glass patch electrodes (with tip resistances of 1–2 MΩ when filled with the internal solution) and connected
to the head stage of an EPC7 patch clamp amplifier (List Electronics, Darmstadt, Germany) or an Axopatch 100A amplifier (Axon
Instruments, Foster City, CA). Voltage clamp and data acquisition and storage were carried out using the Pulse software (r.
7.89; Heka Electronics, Lambrecht/Pfalz, Germany) or pClamp software (Axon Instruments).

Cell capacitance was obtained by integrating the current generated upon a short hyperpolarization from the holding potential
of −70 to −80 mV. The amplitude of the Ca2+ current (ICa) induced by a given depolarization step was determined as the difference between the peak inward current and the current
at the end of the step using Origin (Microcal, Northampton, MA).

Insulin release.

Insulin release from BRIN-BD11 cells in tissue culture was determined using cell monolayers. The BRIN-BD11 cells were harvested
with the aid of trypsin and were seeded into 24-multiwell plates at a density of 5 × 105 per well and were allowed to attach overnight at 37°C. Standard RPMI-1640 tissue culture medium containing 11.1 mmol/l glucose,
which was routinely used to culture the hybrid β-cells, was removed from each well and replaced with culture media containing
1.1, 11.1, and 33.3 mmol/l glucose, respectively. The cells were then incubated for 24 h, and aliquots of the culture media
were removed from each separate well and stored at −20°C for subsequent insulin measurement by radioimmunoassay (12). Insulin output was expressed in terms of the number of cells in each well at the end of the experiment.

Statistics.

The results are expressed as means ± SE. The statistical significance of differences between data were assessed using the
Student’s t test or ANOVA followed by Tukey’s post test.

RESULTS

BRIN-BD11 cells were stably transfected with a mammalian expression vector pcDNA3(−) containing an insert coding for the human
pancreatic β-cell NCX1.7 exchanger (GenBank accession no. AF108388). A clone of cells expressing high levels of Na/Ca exchange
activity (clone 8) was used for studies described here. However, similar results were observed with other clones (3, 4, 5,
and 6).

In a previous study, we identified two NCX1 isoforms (NCX1.3 and NCX1.7) in both rat pancreatic β-cells and RINm5F cells (20). With BRIN-BD11 cells being produced by electrofusion of normal rat pancreatic β-cells with RINm5F cells, we looked for
the presence of these two NCX1 isoforms in BRIN-BD11 cells. Using primers designed to anneal to conserved sequences flanking
the splicing area of NCX1, PCR amplification yielded two bands of 379 and 310 bp (Fig. 1A, lane 2) indicating the presence of exons BDF and BD characterizing NCX1.7 and NCX1.3 isoforms, respectively. In NCX1.7-transfected
cells, PCR amplification also yielded two bands of identical molecular weight, with the upper NCX1.7 transcript being markedly
enhanced (Fig. 1A, lane 3).

On gel electrophoresis, the Na/Ca exchanger migrates as a 120-kDa and 70-kDa protein, with the 120-kDa band being commonly
accepted to represent the mature glycosylated protein and the 70-kDa protein representing a proteolytic fragment of the 120-kDa
band (32). An immunoblot of membranes from control cells (Fig. 1B, lane 1) showed one single clear band at ∼120 kDa. An immunoblot of membranes from NCX1.7-transfected cells (Fig. 1B, lane 2) showed markedly increased amounts of both 120-kDa and 70-kDa bands. The newly expressed 120-kDa band migrated slightly more
slowly than the native one, indicating a slight difference in protein processing. For the purpose of comparison, a comparative
immunoblot of primary rat β-cell membranes and of nontransfected BRIN cells is shown in Fig. 1C. In rat β-cells, the exchanger migrated as a 120-kDa protein, the level of expression of the exchanger being comparable with
that of nontransfected BRIN-BD11 cells.

The overexpression of NCX1.7 protein at the level of the plasma membrane of the transfected cells was visualized by immunofluorescence
using a monoclonal antibody directed against the canine cardiac Na/Ca exchanger (Fig. 2). In these cells (e.g., clone 4 and 8), an intense staining predominantly located at the periphery of cells can be seen (Fig. 2, lower panels), whereas nontransfected cells only displayed a faint fluorescence at the periphery of the cells (Fig. 2, upper and right panel).

To obtain functional evidence of NCX1.7 overexpression, Ca2+ uptake and [Ca2+]i changes induced by extracellular Na+ removal were examined (Figs. 3 and 4). In previous studies, we showed that this maneuver stimulated Ca2+ entry and [Ca2+]i rise in pancreatic β-cells selectively by Na/Ca exchange (reverse mode) (10,11). Figure 3 illustrates the time courses of Na+i-dependent Ca2+ uptake in control and NCX1.7-transfected cells. In both cell types, 45Ca uptake displayed an initial and rapid phase followed by a plateau phase. However, the increase in 45Ca uptake was more rapid and marked in transfected cells than in nontransfected cells. At the plateau phase (120 s), the increase
in uptake was about two to three times larger in transfected cells than in nontransfected cells (P < 0.01). Figure 4 illustrates the effect of extracellular Na+ removal on [Ca2+]i. In control cells, Na+ removal induced a modest and reversible [Ca2+]i increase. In contrast, in NCX1.7-transfected cells, extracellular Na+ removal induced a more rapid, abrupt, and marked increase in [Ca2+]i. Thus, at the peak, the increase in [Ca2+]i was about four to five times larger in transfected cells than in nontransfected cells (P < 0.0001). In this series of experiments, like in others (Figs. 5–7), basal [Ca2+]i did not differ between control and overexpressing cells and averaged 165 ± 8 nmol/l (n = 200).

Taken as a whole, these results support the view that Na/Ca exchange was functionally more active in NCX1.7-overexpressing
cells than in control cells.

To evaluate the role played by Na/Ca exchange in Ca2+ homeostasis, we examined the effect of NCX1.7 exchanger overexpression on the changes in [Ca2+]i induced by membrane depolarization. Figure 5 illustrates the effect of KCl (50 mmol/l) on [Ca2+]i. In control cells, K+ induced a biphasic increase in [Ca2+]i consisting in an initial peak followed by a plateau phase. The increase in [Ca2+]i was rapidly reversible upon removal of K+ from the solution. In NCX1.7-overexpressing cells, several differences could be observed. First, K+ induced a more rapid increase in [Ca2+]i. Thus, the maximum [Ca2+]i was reached after 144 ± 5 and 112 ± 5 s in control and overexpressing cells, respectively (P < 0.0001). Likewise, whereas the rate of [Ca2+]i increase averaged 10.1 ± 0.2 nmol · l−1 · s−1 (n = 94) in control cells, it averaged 17.8 ± 0.3 nmol · l−1 · s−1 in NCX1.7-overexpressing cells (n = 106, P < 0.0001). Second, the [Ca2+]i observed at the steady state (before K+ removal) was higher in transfected cells (725 ± 17 nmol/l) than in control cells (610 ± 15 nmol/l, P < 0.0001). Last, the decrease in [Ca2+]i seen on membrane repolarization was more rapid in overexpressing cells (7.0 ± 0.2 nmol · l−1 · s−1) than in control cells (5.2 ± 0.2 nmol · l−1 · s−1, P < 0.0001) and occurred about 30 s earlier.

Effect of NCX1.7 overexpression on glucose-induced [Ca2+]i increases and oscillations.

To further examine the effect of NCX1.7 exchanger overexpression on Ca2+ handling in BRIN-BD11 cells, [Ca2+]i changes evoked by 11.1 mmol/l glucose were investigated. This concentration of glucose elicits near-maximal stimulation of
insulin secretion from BRIN-BD11 cells (31). Figure 6 illustrates typical individual [Ca2+]i responses induced by glucose in control and NCX1.7-overexpressing cells (Fig. 6A and B, respectively). After a rapid increase in [Ca2+]i, control cells responded to glucose by displaying large amplitude oscillations. In contrast, transfected cells responded
by a delayed and more modest increase in [Ca2+]i with small oscillations, which barely differed from those observed during the baseline. Although BRIN-BD11 cells showed high
variability in their individual response, the mean response of all cells was calculated and is displayed in Fig. 6C. It is clear from the figure that a major difference was that while nontransfected cells showed clear oscillations, no such
oscillations were seen in NCX1.7-transfected cells. At the steady state, the level of fluorescence was slightly lower in overexpressing
cells than in control cells (173 ± 13 vs. 190 ± 14 nmol/l, P > 0.8).

Effect of NCX1.7 overexpression on a tolbutamide-induced increase in [Ca2+]i.

Figure 7 shows typical individual [Ca2+]i responses induced by 100 μmol/l tolbutamide (in the presence of 2.8 mmol/l glucose) (Fig. 7A and B) as well as the mean of the responses seen in control and NCX1.7-transfected cells (Fig. 7C). Like in the case of glucose, nontransfected cells displayed clear oscillations; overexpressing cells did not. Again in
Fig. 7C, it can be seen that although control cells showed clear oscillations, the transfected cells showed a staircase increase
in [Ca2+]i without such distinct oscillations.

Effect of NCX1.7 overexpression on voltage-sensitive ICa.

To exclude any influence of NCX1.7 overexpression on voltage-sensitive ICa, ICa was measured using the patch clamp technique. The holding potential was set at −70 mV, and the command voltage consisted
of steps, given every 3 min, from the holding potential to a preconditioning level at −40 mV for 0.5 s (to inactivate voltage-dependent
Na+ currents), followed by a 6- to 8-s depolarization to 0 mV to activate the L-type Ca2+ current. Round cells were used in both groups and were of the same size (membrane capacitance 17.7 ± 1.0 vs. 19.2 ± 1.6 pF
in control and transfected cells, respectively). Gigaohm seal resistances were also similar (4.0 ± 1.1 vs. 3.5 ± 0.6 GΩ in
control and transfected cells, respectively). The current at 0 mV could be suppressed by nifedipine (10 μmol/l), indicating
that it was due to L-type Ca2+ channels. Our preliminary data (not presented) showed that ICa inactivates over a few seconds and recovers from inactivation slowly. Figure 8A illustrates typical recordings obtained in a control and in a transfected cell. Both currents decayed from their peak value,
with a half-time of 724 and 868 ms, respectively. Pooled data of peak current amplitudes and half-time of decay from nine
control and nine transfected cells are given in Fig. 8B and C, respectively. Although the current amplitude tended to be larger in transfected cells (−8.00 ± 1.7 pA/pF), the difference
with control cells (−5.7 ± 0.8 pA/pF) did not reach statistical significance (P > 0.20). Similarly, there was no significant difference between the half-time of inactivation between the two groups of cells
(1,051 ± 158 vs. 1,000 ± 178 ms in control and transfected cells, respectively).

Effect of diazoxide on glucose-induced Ca2+ oscillations in NCX1.7-overexpressing cells.

If the absence of large amplitude [Ca2+]i oscillations in overexpressing cells results from an increased Na/Ca exchange depolarizing current, then this absence should
be reversed by membrane hyperpolarization. Figure 9 shows that this is indeed the case. Thus, diazoxide (2.5–10 μmol/l), which hyperpolarizes the plasma membrane by opening
ATP-dependent K+ channels (33), restored glucose-induced large amplitude [Ca2+]i oscillations in NCX1.7-overexpressing cells.

Effect of NCX1.7 overexpression on 45Ca uptake.

45Ca uptake was measured over 90 min of incubation in the presence of glucose (1.1 and 11.1 mmol/l, Fig. 10A). In nontransfected cells, glucose induced a modest increase in 45Ca uptake. In overexpressing cells, basal 45Ca uptake (at 1.1 mmol/l glucose) was about 100% higher than that in nontransfected cells (P < 0.001), and glucose induced a six times larger increase in 45Ca uptake than in nontransfected cells (P < 0.001).

Effect of NCX1.7 overexpression on glucose-induced insulin secretion.

Insulin release was measured over a 24-h static incubation in RPMI-1640 tissue culture medium in the presence of 1.1 or 11.1
mmol/l glucose. In nontransfected cells, 11.1 mmol/l glucose induced a two- to threefold increase in insulin release (P < 0.05, Fig. 10B). In NCX1.7 transfected cells, basal insulin release, measured at 1.1 mmol/l glucose, was markedly increased (P < 0.001), and the increase in insulin release induced by 11.1 mmol/l glucose was about 10 times larger than that in nontransfected
cells (P < 0.01, Fig. 10B). To examine the extent to which this increase in insulin release could be related to the overexpression of the exchanger,
the effect of glucose on insulin release was examined in four other clones (clones 3–6). However, none of the latter clones
showed an increase in the insulinotropic action of glucose, suggesting that the potentiating effect on glucose-induced insulin
release seen in clone 8 was not due to Na/Ca exchanger overexpression.

DISCUSSION

In the present work, we successfully and stably overexpressed one of the pancreatic β-cell Na/Ca exchanger splice variants
(NCX1.7) in insulin-producing BRIN-BD11 cells. The overexpression could be assessed at both the mRNA and protein level. The
overexpressed protein was appropriately targeted to the plasma membrane, as shown by microfluorescence but also by the increase
in Na/Ca exchange activity observed in transfected cells.

The Na/Ca exchanger works predominantly as a Ca2+ extrusion mechanism (forward mode) but may also reverse (reverse mode) under physiological conditions, for example, upon
membrane depolarization or in response to an increase in intracellular Na+ concentration (13,34). The present data clearly show that both modes of activity were increased in NCX1.7-overexpressing cells. The increase in
forward mode activity was best evidenced in K+ experiments, where the decrease in [Ca2+]i seen on membrane repolarization was about 30 s earlier and faster in overexpressing than in control cells. The increase in
reverse Na/Ca exchange activity of our clone was also obvious, with the rise in 45Ca uptake and [Ca2+]i induced by the removal of extracellular Na+ being more marked in overexpressing cells than in control cells. This increase in reverse Na/Ca exchange activity was also
evident from the K+ experiments, in which a more rapid rise (about twofold) in [Ca2+]i was observed on membrane depolarization. Because the Ca2+ current was unchanged in overexpressing cells compared with control cells, the accelerated rise in [Ca2+]i can be attributed to an increase in reverse Na/Ca exchange activity. Likewise, the higher [Ca2+]i recorded at the steady state in the same experiments can also be taken as evidence for increased reverse Na/Ca exchange activity.
In previous studies, an increase in both forward and reverse Na/Ca exchange activity was also observed in cardiomyocytes overexpressing
the Na/Ca exchanger (35–37).

The main interest of the present work was to show the functional consequences of Na/Ca exchanger overexpression and to determine
whether the level of Na/Ca exchange activity may shape stimulus-induced cytosolic Ca2+ oscillations. First, Na/Ca exchanger overexpression did not affect basal [Ca2+]i, a finding compatible with the view that Na/Ca exchange displays a low affinity but high capacity for Ca2+ and that it does not participate in the fine tuning of [Ca2+]i around the basal level (5). In no other studies of Na/Ca overexpression could a change in basal [Ca2+]i be observed (35–37). Second, Na/Ca overexpression increased Ca2+ entry and the rise in [Ca2+]i in response to membrane depolarization (Fig. 4 and 5). Likewise, it accelerated the decrease in [Ca2+]i due to membrane repolarization (Fig. 5). This result indicates that within the range of the changes in [Ca2+]i provoked by K+ (150–1,000 nmol/l), Na/Ca exchange plays a significant role in the rat β-cell [Ca2+]i homeostasis, with the exchanger being a versatile system allowing both Ca2+ entry and outflow. In agreement with such a view, Na/Ca exchanger overexpression was associated with an increase in 45Ca uptake whether at low or high glucose concentrations. Because this increase was seen at both glucose concentrations, it
probably results from a higher 45Ca2+ turnover rate leading to a faster access to radioisotopic equilibrium. It constitutes a further proof for an increased forward
and reverse Na/Ca exchange activity in our transfected cells and indicates that glucose activates these two modes. Indeed,
the difference in 45Ca uptake between control and overexpressing cells was more marked at the high concentration than at the low concentration
of glucose.

The present data are complementary to those obtained in rat β-cells treated with antisense oligonucleotides to knock down
the expression of the exchanger. In the latter study, we observed a modest but significant reduction in the rise in [Ca2+]i induced by membrane depolarization together with a major slowdown (70%) of the Ca2+ removal rate from the cytoplasm upon membrane repolarization (13). Taken together, these data constitute a strong indication for the prominent role played by Na/Ca exchange in its two modes
of function in [Ca2+]i homeostasis in rat pancreatic β-cells.

The aim of the present work was to examine the extent to which an increase in Na/Ca exchange activity could shape the electrical
activity of the pancreatic β-cell and explain the differences in glucose-induced electrical activity and [Ca2+]i oscillations in mouse and rat β-cells. Indeed, we previously suggested that the inward current generated by the exchange
prolongs the duration of the burst of electrical activity induced by glucose, namely transforms an oscillatory pattern into
continuous electrical activity. As expected, overexpression of the Na/Ca exchanger suppressed the large oscillations induced
by glucose and replaced them with a staircase increase in [Ca2+]i onto which oscillations of small amplitude were superimposed. Such a change was observed also when tolbutamide was used to
stimulate the cells. Therefore, the difference in Na/Ca exchanger level of expression and activity (22) between the mouse and the rat β-cell may indeed explain the difference in electrical and [Ca2+]i behavior between the β-cells of these two species. Such a view was confirmed by the demonstration that a slight hyperpolarization
of the plasma membrane induced by a low concentration of diazoxide restored large amplitude [Ca2+]i oscillations in NCX1.7-overexpressing cells.

Incidentally, Na/Ca exchanger knockout using antisense oligonucleotides also led to a decrease in the Ca2+ oscillations induced by glucose and tolbutamide (13). However, when considering the present data and those obtained with antisense oligonucleotides, it must be kept in mind
that the Na/Ca exchanger is a complex system that allows both Ca2+ entry and outflow and generates in addition an inward current. In comparison, the plasma membrane Ca2+-ATPase only extrudes Ca2+. Hence, with Na/Ca exchange, one may not necessarily expect opposite effects on repressing and overexpressing the exchanger.
For instance, in the present study, overexpression of the exchanger influences the oscillations induced by glucose and tolbutamide
mainly by generating an inward current that prolongs the duration of the burst of electrical activity, namely transforming
an oscillatory pattern into a continuous electrical activity, with parallel changes in [Ca2+]i. When Na/Ca exchanger is repressed by antisense oligonucleotides, the oscillations may persist (no Na/Ca exchange inward
current) but could be altered by a reduction in Ca2+ inflow and outflow.

It has been shown that the ability of mouse β-cells to display large [Ca2+]i oscillations is closely related to their handling of Na+ (29). For instance, amino acids cotransported with Na+ have been found to transform the slow [Ca2+]i oscillations induced by glucose into a sustained increase. Likewise, inhibition of the Na+/K+ pump by the removal of extracellular K+ or addition of ouabain leads to a similar transformation (29). Therefore, the β-cell of the rat, by having a higher rate of Na/Ca exchange activity than that of the mouse, differs from
the latter in a similar respect (Na+ handling), explaining the differences in glucose-induced electrical activity and [Ca2+]i oscillations between these cells. Thus, a higher rate of Na/Ca exchange would lead to a higher rate of Na+ entry and increase in cytosolic Na+ concentration similar to that obtained with ouabain and amino acids cotransported with Na+. Furthermore, the rapidly descending phases of mouse β-cell [Ca2+]i oscillations have been proposed to result from the outward transport of Ca2+i from the cell, a finding compatible with the view that glucose stimulates such an outward transport (29). Consequently, it can be expected that enhanced removal of Ca2+ from the cytoplasm would prevent the establishment of the critical [Ca2+]i required for a feedback inhibition of a glucose-stimulated entry of the ion (29). Such a view is consistent with the observation that the acceleration of the outward transport of Ca2+ by protein kinase activation suppresses the slow [Ca2+]i oscillations seen in mice (29). Such a mechanism might explain why the β-cell of the rat, which shows a higher rate of forward Na/Ca exchange activity
than that of the mouse and hence a higher rate of Ca2+ extrusion, display less slow [Ca2+]i oscillations than the β-cell of the mouse (13). Human β-cells also display [Ca2+]i oscillations in response to glucose (29), which, however, appear to be closer to those of rat β-cells than to those of mouse β-cells. It would be of interest to
measure the activity of the exchanger in such human β-cells.

In conclusion, the present data confirm that Na/Ca exchange plays a significant role in the rat β-cell [Ca2+]i homeostasis and show that the current generated by the exchanger appears to shape stimulus-induced membrane potential and
[Ca2+]i oscillations in insulin-secreting cells, with the difference in electrical activity and [Ca2+]i behavior seen in mouse and rat β-cells resulting in part from a difference in Na/Ca exchange activity between these two cells.

A: Reverse-transcribed PCR amplification of NCX1 in control and NCX1.7-transfected BRIN-BD11 cells. Specific primers designed
to anneal to conserved sequences flanking the splicing area were used. Lane 1: Molecular weight marker (1-kb DNA ladder; sizes in bases). Lane 2: Control cells. Lane 3: NCX1.7-transfected cells. The PCR products were separated by agarose gel (1.5%) electrophoresis and stained with ethidium
bromide. B: Western blot analysis of NCX1 splice variants expressed in control and NCX1.7-transfected BRIN-BD11 cells. Lane 1: Control cells. Lane 2: BRIN-BD11 cells transfected with human NCX1.7. The lanes were loaded with 10 μg protein. The positions and sizes of the
molecular mass standards are indicated on the right and expressed in kilodaltons. The positions of the so-called 120-kDa and
70-kDa proteins are also shown on the left. C: Western blot analysis of NCX1 splice variants expressed in control and rat primary β-cells. Lane 1: Control BRIN-BD11 cells. Lane 2: Rat primary β-cells. The lanes were loaded with 10 μg protein. The positions and sizes of the molecular mass standards are
indicated on the right and expressed in kilodaltons. The position of the so-called 120-kD protein is also shown on the left.

Indirect immunofluorescence microscope images of BRIN-BD11 cells. Top panels: nontransfected cells. Lower panels: two different
clones of NCX1.7 transfected cells (clones 4 and 8). The nuclei of the cells are labeled in blue (DAPI). In the upper right
and the two lower panels, Na/Ca exchanger protein was immunostained using a mouse monoclonal anti-canine Na/Ca exchanger antibody
and revealed by a secondary antibody (red color).

45Ca uptake by reverse Na/Ca exchange in control and NCX1.7-transfected BRIN-BD11 cells. Na/Ca exchange was measured as intracellular
Na+-dependent 45Cao uptake. The time courses of 45Ca uptake were measured in control (•) and NCX1.7-transfected (▪) cells after reversal of Na/Ca exchange induced by Na+o (extracellular Na+) removal. Means ± SE refer to four to six individual experiments, comprising six replicates in each case.

Effect of extracellular Na+ removal on [Ca2+]i in control and NCX1.7-transfected BRIN-BD11 cells. The period of exposure to Na+-free medium is indicated by a bar above the curves. The curves shown are the mean of 77 and 208 traces, respectively.

Effect of KCl on [Ca2+]i in control and NCX1.7-overexpressing BRIN-BD11 cells. The bar above curves indicates the period of KCl exposure. The curves
shown are the mean of 94 and 106 traces, respectively.

Effect of 11.1 mmol/l glucose on [Ca2+]i in control and NCX1.7-overexpressing BRIN-BD11 cells. A and B show typical individual traces observed in control and NCX1.7-transfected cells, respectively. The curves shown in C are the mean of 87 and 84 traces, respectively. Periods of exposure to glucose are indicated by the bars above the curves.

Effect of 100 μmol/l tolbutamide in the presence of 2.8 mmol/l glucose on [Ca2+]i in control and NCX1.7-overexpressing BRIN-BD11 cells. A and B show typical individual traces observed in control and NCX1.7-transfected cells, respectively. The curves shown in C are the mean of 37 and 54 traces, respectively. Periods of exposure to tolbutamide are indicated by the bars above the curves.

Whole-cell Ca2+ currents recorded in the ruptured patch configuration in response to 6–8 s depolarization to 0 mV from holding potential
of −70 mV and a preconditioning level of −40 mV. A: Typical recordings obtained in control and transfected cells. B and C: Pooled data of peak current amplitudes (B) and half-time of decay (C) from nine control and nine transfected cells. ICa-L, Ca2+ current through L-type Ca2+ channels.

Effect of glucose (11.1 mmol/l) and diazoxide (5 μmol/l) on [Ca2+]i in NCX1.7-overexpressing cells. The figure shows a typical individual trace among 48 traces (four coverslips, two preparations,
and a similar number of experiments being carried out with 2.5 and 10 μmol/l diazoxide and giving similar results).

A: Effect of glucose on 45Ca uptake in nontransfected (NT; ▪) and NCX1.7-transfected BRIN-BD11 (□) cells. Means ± SE refer to four individual experiments
comprising six replicates in each case. B: Effect of glucose (11.1 mmol/l) on insulin release from control (▪) and NCX1.7-overexpressing (□) cells over 24 h of incubation.
Mean ± SE refer to four individual experiments comprising four replicates in each case.